title: Monothiol glutaredoxin cDNA from Taiwanofungus camphorata: a novel CGFS-type glutaredoxin possessing glutathione reductase activity.
abstract: Glutaredoxins (Grxs) play important roles in the redox system via reduced glutathione as a reductant. A TcmonoGrx cDNA (1039 bp, EU158772) encoding a putative monothiol Grx was cloned from Taiwanofungus camphorata (formerly named Antrodia camphorata). The deduced amino acid sequence is conserved among the reported monothiol Grxs. Two 3-D homology structures of the TcmonoGrx based on known structures of human Grx3 (pdb: 2DIY_A) and Mus musculus Grx3 (pdb: 1WIK_A) have been created. To characterize the TcmonoGrx protein, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli C41(DE3). The recombinant His6-tagged TcmonoGrx was overexpressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed a predominant band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited glutathione reductase (GR) activity via dithionitrobenzoate (DTNB) assay. The Michaelis constant (K(M)) values for GSSG and NADPH were 0.064 and 0.041 mM, respectively. The enzyme's half-life of deactivation at 60 °C was 10.5 min, and its thermal inactivation rate constant (k(d)) was 5.37 × 10(-2) min(-1). The enzyme was active under a broad pH range from 6 to 8. The enzyme retained 50% activity after trypsin digestion at 37 °C for 40 min. Both mutants C(40)→S(40) and C(165)→S(165) lost 40-50% GR activity, whereas the mutant S(168)→C(168) showed a 20% increase in its GR activity.
authors.affiliation: Institute of Biotechnology, National Changhua University of Education, Changhua, Taiwan.
authors.forename: Chuian-Fu
authors.initials: CF
authors.lastname: Ken
authors.forename: I-Jing
authors.initials: IJ
authors.lastname: Chen
authors.forename: Chao-Ting
authors.initials: CT
authors.lastname: Lin
authors.forename: Shiu-Mei
authors.initials: SM
authors.lastname: Liu
authors.forename: Lisa
authors.initials: L
authors.lastname: Wen
authors.forename: Chi-Tsai
authors.initials: CT
authors.lastname: Lin
mesh_terms: D000363:Agaricales / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D054477:Glutaredoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005980:Glutathione Reductase / Q000737:chemistry / Q000378:metabolism*; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D054477:Glutaredoxins; D005980:Glutathione Reductase